Stem cell transplantation has great clinical potential for treating many conditions, but toxicities associated with conditioning and graft vs. host disease limit its application. Innovative protocols based on costimulation blockade suggest that these toxicities can be overcome. However, we have discovered that virus infection at the time of bone marrow transplantation into sub-lethally irradiated mice treated with costimulation blockade prevents marrow engraftment and leads to the death of the host. The goals of this Project are: 1) minimizing hazardous conditioning regimens and 2) discovering how virus infection compromises hematopoietic stem cell engraftment and recipient safety. This project is based the hypothesis that establishment of allogeneic hematopoietic chimerism and generation of donor-specific central tolerance depends on the induction of peripheral transplantation tolerance. This hypothesis predicts that mechanisms by which virus infection prevents hematopoietic stem cell engraftment will be similar to those that compromise allograft survival in peripheral tolerance protocols. Testing this hypothesis, we have already made the exciting discovery that treatment with a donor-specific transfusion (DST) plus anti-CD 154 mAb (a peripheral tolerance protocol) deletes host alloreactive CD8+ T cells and permits the engraftment of allogeneic bone marrow in the complete absence of any myeloablative conditioning. To move this discovery forward, we propose 3 Aims. Aim 1 is to test the hypothesis that virus infection matures APCs to provide bystander activation and prevent the deletion of host CD8 + cells and bone marrow engraftment in mice treated with DSTplus anti-CD154 mAb. Aim 2 is to test the hypothesis that virus infection at the time of transplantation prevents engraftment of bone marrow cells by maturing dendritic cells and preventing the generation of regulatory cells. Aim 3 is to test the hypothesis that allo-memory CD8+ T cells in virus-immune mice prevent the engraftment of allogeneic bone marrow. To test these hypotheses, we will use two innovative technologies: 1) techniques in the third project and the Virology Core for identifying virus-immune T cells and determining their antigen-specificity at the single cell level. 2) a new "synchimera" model based on TCR transgenic mice to quantify alloreactive T cells. Our long term goal is the translation of this new approach for stem cell transplantation and central tolerance induction into a clinically powerful and safe form of therapy for human disease.